PROJECT SUMMARY The most common methods to study the human microbiome have involved descriptive studies of bacterial DNA (metagenomics), RNA (transcriptomics), metabolites (metabolomics) or proteins (proteomics) in patient cohorts. These ?omics? studies have established a strong link between the microbiome and metabolic diseases such as diabetes and obesity. Unfortunately, little is known about the mechanisms through which commensal bacteria affect metabolic pathways (i.e. effector functions). Without knowledge of how bacterial DNA and its encoded proteins/metabolites translate into changes in human biology, ?omics? studies are limited to correlative observations and not mechanisms that define the relationship between microbes and human health. We propose a shift from descriptive ?omics? studies to functional studies of the microbiome to isolate microbial effectors that shape metabolic outcomes and improve our understanding of disease pathophysiology and treatment. It is increasingly clear that the microbiome interacts with human cellular pathways through G-protein- coupled receptors (GPCRs). As GPCRs are linked to the pathophysiology of metabolic diseases and their treatment, the identification of GPCR-active microbial effectors presents a strategy to understand how the microbiome impacts metabolic disease outcomes and to develop microbiome-based therapies (i.e. live biotherapeutics). In previous work as part of my K08 award, we used functional metagenomics to isolate a family of bacterial genes that encode for GPCR-active small molecules. One GPCR-active small molecule we identified, N-acyl serinol (N-AS), is a GPR119 agonist. GPR119 is a metabolic GPCR that is a therapeutic target for the treatment of diabetes and obesity as GPR119 regulates GLP-1 and insulin release. We demonstrated that bacteria engineered to produce GPR119-active metabolites can in wild-type mice regulate insulin, GLP-1 and glucose homeostasis similar to GPR119 agonists in clinical trials. This data supports that the identification of microbial effectors in vitro informs our understanding of microbiome physiology in vivo and these interactions can be developed therapeutically. We have since expanded our methods from screening of bacterial DNA to identify effector genes (K08) to screening of bacterial culture broths to identify GPCR-active metabolites (functional metabolomics). This work suggests bacteria affect diverse metabolic GPCR beyond GPR119. The central hypothesis is that functional interrogation of bacterial culture broths to identify GPCR-active metabolites (functional metabolomics) will improve our understanding of metabolic disease pathophysiology and identify novel therapeutic strategies. We will develop this hypothesis by complementing previous methods to isolate bacterial effector genes (K08) with new methods to isolate effector metabolites (Aim 1) from bacteria enriched or depleted in patients with metabolic diseases. We will also explore the role of N-AS and GPR119 in mouse disease models (Aim 2) to understand their role in metabolic disease pathophysiology and treatment.